Dual-isotope (DI) studies offer a number of advantages in pre-clinical imaging. These include: reducing study times when compared with sequential scans, reducing the number of animals required for any given study, and most importantly, producing images perfectly registered in space and time that provide simultaneous information about two distinct body functions. The ability of single photon emission computed tomography (SPECT) to measure and differentiate energies of the emitted photons makes it well suited for DI imaging. However, since scattered photons originating from one radioisotope may be detected in the energy window of the other and thus degrade image quality and quantitative accuracy, scatter and crosstalk corrections must be applied. The decay characteristics of 111In and 67Ga, which are suitable for quantitative DI imaging for up to 2 weeks post-injection, led us to investigate the performance of simultaneous 111In/67Ga SPECT imaging using a small-animal pre-clinical scanner. A series of phantom experiments were performed to investigate image quality and accuracy of activity quantification in 111In/67Ga images acquired with three different collimators and reconstructed from different photopeak combinations. The triple energy window (TEW) method was used to correct for scatter and crosstalk. Based on these phantom studies, the optimal selection of collimator and energy window settings was determined. When using these optimal settings, submillimeter-size structures were distinguishable in the reconstructed images and quantification errors below 20% were achieved for both isotopes. The optimal parameters were subsequently applied to an in vivo animal study. The determination of the distinct pharmacokinetic profiles of two polymer radiopharmaceuticals injected simultaneously, but by different administration routes (intravenously and intraperitoneally) into a single animal demonstrated the feasibility of simultaneous 111In/67Ga SPECT.